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  Volume 12(4) Hypothesis

Prediction driven functional annotation of hypothetical proteins in the major facilitator superfamily of S. aureus NCTC 8325 Jessica Marklevitz1, Laura K. Harris1*

1Department of Science, Davenport University, 200 S. Grand Ave, Lansing, MI, 48933 United States of America *Corresponding author: laura.harris@davenport.edu Received June 26, 2016; Revised July 10, 2016; Accepted July 11, 2016; Published July 26, 2016 Abstract: Antibiotic resistance Staphylococcus aureus strains cause several life threatening infections. New drug treatment options are needed, but are slow to develop because 50% of the S. aureus genome is hypothetical. The goal of this is to aid in the annotation of the S. aureus NCTC 8325 genome by identifying hypothetical proteins related to the Major Facilitator Superfamily (MFS). The MFS is a broad protein group with members involved in drug efflux mechanisms causing resistance. To do this, sequences for three MFS proteins with x-ray crystal structures in E. coli were PSI-BLASTed against the S. aureus NCTC 8325 genome to identify homologs. Eleven identified hypothetical protein homologs underwent BLASTP against the non-redundant NCBI database to fit homologs specific to each hypothetical protein. ExPASy characterized the physiochemical features, CDD-BLAST and Pfam identified domains, and the SOSUI server defined transmembrane helices of each hypothetical protein. Based on size (300 – 700 amino acids), number of transmembrane helices (>7), CD06174 and MFS domains in CDD-BLAST and Pfam, respectively, and close relation to well-defined homologs, SAOUHSC_00058, SAOUHSC_00078, SAOUHSC_00952, SAOUHSC_02435, SAOUHSC_02752, and ABD31642.1 are members of the MFS. Further multiple-alignment and phylogeny analyses show SAOUHSC_00058 to be a quinolone resistance protein (NorB), SAOUHSC_00058 a siderophore biosynthesis protein (SbnD), SAOUHSC_00952 a glycolipid permease (LtaA), SAOUHSC_02435 a macrolide MFS transporter, SAOUHSC_02752 a chloramphenicol resistance (DHA1), and ABD31642.1 is a Bcr/CflA family drug resistance efflux transporter. These findings provide better annotation for the existing genome, and identify proteins related to antibiotic resistance in S. aureus NCTC 8325.

Background: Staphylococcus aureus is an opportunistic pathogen responsible for a wide variety of infections including superficial skin and surgical wound infections, toxic shock syndrome, and bacteremia [1]. Most are nosocomial infections, though there are increases in community acquired (CA) Methicillin-resistant Staphylococcus aureus (MRSA) infections, particularly among immunocompromised patients. Other health issues related to internalized infections are heart and lung diseases such as endocarditis and necrotizing pneumonia found in younger community populations rather than remaining solely a hospital acquired infection. Deaths from S. aureus caused heart and lung infections are reported [2]. In 2011, the Center for

Disease Control estimate 80,000 invasive MRSA infections and 11,285 related deaths in the United States [3]. These deaths are primarily due to MRSA strains that are resistant to macrolides, monovalent cationic antimicrobials, quinolones, bivalent quaternary ammonium compounds, tetracycline, and all beta-lactam antibiotics including penicillin, amoxicillin, methicillin, and oxacillin [4]. Inactivation of antibiotics, reduction in cellular permeability, alteration of antibiotic target sites, and bacterial efflux pumps convey drug resistance [5]. Several multidrug efflux genes, such as the NorA, NorB, and NorC from the S. aureus chromosome, confer resistance to quinolones and other antibiotics [6, 7]. Disturbingly, an increase in the variety of drug-resistant strains of

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S. aureus has been noted in the past years, with the most prevalent being vancomycin-resistant S. aureus (VRSA). Usually VRSA develops in MRSA patients treated with vancomycin, the frontline treatment to MRSA. While VRSA is rare with most S. aureus being vancomycin-intermediate meaning that large amounts of vancomycin still kill the organisms, this presents a new challenge to combat S. aureus infections. These superbugs are generally sensitive to intravenous medication, such as quinupristin–

dalfopristin, that require slow infusion in a large fluid volume, making it unrealistic for administration to CA-MRSA patients in an outpatient setting. Quinupristin–dalfopristin can also cause disabling myopathy as a side effect. Due to documented increases of a global spread of CA-MRSA in just the past 20 years and the increases in antibiotic resistances, there is a need for new treatment options [2].

Figure 1: Experimental Overview. Protein sequences for three E. coli MFS proteins underwent PSI-BLAST. Hypothetical protein homologs meeting five parameters are likely functional MFS and then compared to homologs of predicted function.

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Most multidrug resistance efflux pumps eject various substrates regardless of structure, a common feature of the Major Facilitator Superfamily (MFS) they belong to [8]. In bacteria, about 25% of characterized membrane transport proteins come from the MFS [9]. This group of transporters contains 74 families that move a wide variety of substrates including sugar phosphates, nucleosides, ions, amino acids, peptides, and drugs across the cytoplasmic membrane [10]. X-ray crystallography established E. coli’s structure of three members of this conserved protein family: glycerol-3-phosphate transporter (GlpT), lactose permease (LacY), and multidrug transporter (EmrD). These structures, listed in the PDB as 1PW4, 1PV7, and 2GFP for GlpT, LacY, and EmrD, respectively, demonstrate that MFS proteins function via the substrate’s electrochemical potential. MFS proteins are usually 400 to 600 amino acids long, with most containing 11 to 14 transmembrane alpha helices connected by hydrophilic loops [10]. Hypothetical proteins make up approximately 50% of reference strain S. aureus NCTC 8325 genome [11]. Nucleic acid sequence only predicts hypothetical proteins [12]. There is no experimental evidence for a hypothetical protein’s function exists; ergo a hypothetical protein may not actually be functional. Further, some hypothetical proteins do not follow conventional phylogenetic lineage. Usually the two groups of hypothetical proteins are uncharacterized protein families and domains of unknown function, or experimentally identified proteins with structural domains that do not relate to already established functions. However, databases frequently label a protein hypothetical if it comes from a newly deposited sequence and no annotation was available for it at that time [13]. This is likely the case with S. aureus NCTC 8325, whose genome was published in 2006 [14].

With approximately half of all S. aureus NCTC 8325 genomic protein sequences currently annotated as hypothetical proteins and 25% of all membrane transport proteins belonging to the MFS, ergo likely related to antibiotic resistance, there is great potential for the discovery of new drug targets here [15]. Since proper annotation of hypothetical proteins can lead to new therapeutic targets, a high demand to characterize hypothetical proteins is present. Ergo, this study uses in silico techniques to identify and characterize hypothetical proteins in S. aureus NCTC 8325 that are related to the protein MFS. Methodology: Figure 1 illustrates the overall experimental design. To identify MFS-related hypothetical proteins, protein sequences for the three E. coli MFS crystal structures, 1PW4 (GlpT), 1PV7 (LacY), and 2GFP (EmrD), were downloaded from PDB. A Position-Specific Iterative Basic Local Alignment Search Tool (PSI-BLAST) revealed proteins of interest when searched with each sequence against the S. aureus NCTC 8325 genome. If the protein was hypothetical, it underwent

protein-protein BLAST (BLASTP) against NCBI’s entire non-reductant protein database to identify top matching homologs in any species. NCBI provides both BLAST algorithms. Table 1: Homologs in S. aureus NCTC 8325 to GlpT from E. coli

Protein Accession QC % %ID

glycerol-3-phosphate transporter WP_001010111.1 97 57 antiporter WP_001008722.1 96 32 glucarate transporter WP_000660709.1 90 22 quinolone resistance protein NorB WP_000381270.1 21 28 multidrug efflux MFS transporter NorA WP_001041272.1 15 27 MFS transporter WP_001154220.1 27 20 probable glycolipid permease LtaA Q2FZP8.2 27 20 conserved hypothetical protein ABD31816.1 7 27 hypothetical protein SAOUHSC_00058 YP_498663.1 56 23 hypothetical protein SAOUHSC_00078 YP_498678.1 50 22 hypothetical protein SAOUHSC_00952 YP_499505.1 41 20 hypothetical protein SAOUHSC_02620 WP_000436818.1 4 45

QC, Query Cover; %ID, percentage of identity; MFS, major facilitator super-family Physicochemical Characterization To characterize the proteins, the Expasy Protparam server computed several physicochemical characterizations. The number of amino acids, molecular weight, total number of charged residues (the addition of arginine and lysine for negatively charged and aspartic acid added to glutamic acid for positively charged) [16]. The algorithm determines the theoretical isoelectric point, the pH where a molecule carries no net electrical charge, from the number of charged residues. Further, the program calculates the extinction coefficient, the amount of light absorbed by the protein at a 280nm wavelength, which is helpful for protein purification procedures [17]. A protein’s stability in a test tube under physiological conditions is measured by its instability index [18]. The relative volume occupied by open side chain amino acids in a protein is the aliphatic index [19]. The grand average hydropathy (GRAVY) is the total of the hydropathy values of all amino acids in the protein divided by the number of resides a measure of hydrophobicity for a given molecule [20]. The SOSUI server also determines a protein’s hydrophobicity, though via solubility computations, and it further characterizes potential transmembrane regions [21]. Domain Identification Both Pfam and the conserved domain database (CDD) identified domains. The Pfam database is a collection of protein families represented by hidden Markov models and multiple sequence alignments [22, 23]. Conserved domain database BLAST (CDD-BLAST) uses a variant of PSI-BLAST to scan a set of pre-calculated position-specific scoring matrices from a protein query [24]. Researchers frequently use Pfam and CDD-BLAST together to examine protein domains to predict function prior to modeling the protein to evaluate its binding capability [15, 25].

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Protein relatedness Hypothetical proteins between 300 and 700 amino acids long with well-defined BLASTP homologs, CD06174 MFS domain identified by CDD-BLAST, and an MFS domain found by Pfam underwent evolutionary analyses against non-redundant defined homologs identified from prior PSI-BLAST and BLASTP searches. Two programs, PROMALS3D and CLUSTALW, did multiple sequence alignment. PROMALS3D aligned each set of proteins based on homology to the E. coli protein with the established structure by constructing alignments using information from available homologs with 3D structures, sequence database searches, and secondary structure prediction [26]. CLUSTALW aligned all protein sequences together as one large dataset [27]. The PHYLIP package Protdist program used the output from CLUSTALW to produce a distance matrix using default settings such as the Jones-Taylor-Thornton matrix distance model [28]. Another program in

the PHYLIP suite, Neighbor, used this matrix to construct a neighbor joining and unweighted pair group method with arithmetic mean trees. Again as verification, another phylogenetic tree building approach, the Fitch-Margoliash and Least-Squares Distance method, verified the results. DrawTree from the PHYLIP suite illustrated phylogenetic trees produced. All analyses used default settings. Discussion: This study aimed to identify and characterize hypothetical proteins in S. aureus NCTC 8325 that belong to the MFS of proteins. Three E. coli MFS proteins with established structure (GlpT, LacY, and EmrD) underwent PSI-BLAST against the S. aureus NCTC 8325 genome to identify homologs. Tables 1 to 3 list homologs for GlpT, LacY, and EmrD, respectively. This method identified eleven hypothetical proteins. Researchers need further examination to determine if these hypothetical proteins belong to the MFS.

Figure 2: Alignment of GlpT homologs aligned by PROMALS3D. Magenta names are representative sequences colored red to identify predicted alpha-helix secondary structures. The black names belonging to the same alignment group as the magenta name above it, indicating a strong relationship between the two. Consensus_aa, consensus amino acid sequence; Consensus_ss, consensus predicted secondary structures; h, consensus predicted secondary structure alpha-helix.

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Table 2: Homologs in S. aureus NCTC 8325 to LacY from E. coli Protein Accession QC % %ID

multidrug efflux MFS transporter NorA WP_001041272.1 45 28 hydroxyethylthiazole kinase WP_000610051.1 17 25

siderophore biosynthesis protein SbnD WP_000610051.1 17 25

MFS transporter WP_000610884.1 35 27 glucarate transporter WP_000660709.1 46 29 proline/betaine transporter WP_000347061.1 14 27 nitrate transporter NarT WP_000278558.1 17 32 nickel ABC transporter permease WP_000584765.1 19 34 antibiotic MFS transporter WP_000675401.1 11 29 hypothetical protein SAOUHSC_02866 YP_501322.1 12 31 hypothetical protein SAOUHSC_02307 YP_500786.1 23 23 hypothetical protein SAOUHSC_02309 WP_001287088.1 11 31

QC, Query Cover; %ID, percentage of identity; MFS, major facilitator super-family

To distinguish that, homolog identification, physiochemical characterization, transmembrane enumeration, and domain identification compared hypothetical proteins to established MFS proteins. Table 4 lists the top BLASTP homolog for each hypothetical protein regardless of origin. A hypothetical protein that has a homolog with a well-defined function is more likely related. SAOUHSC_02307, SAOUHSC_02309, and ABD31816.1 hit several general membrane proteins. SAOUHSC_02620 and SAOUHSC_02866 matched several hypothetical proteins as well as general membrane proteins. This indicates these hypothetical proteins may not be in the MFS.

Figure 3: Alignment of EmrD homologs aligned by PROMALS3D. Magenta names are representative sequences colored red to identify predicted alpha-helix secondary structures. The black names belonging to the same alignment group as the magenta name above it, indicating a strong relationship between the two. Consensus_aa, consensus amino acid sequence; Consensus_ss, consensus predicted secondary structures; h, consensus predicted secondary structure alpha-helix.

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Table 3: Homologs in S. aureus NCTC 8325 to EmrD from E. coli Table 4: Homologs to hypothetical proteins Protein Accession QC % %ID Hypothetical Protein Top Match

Bcr/CflA family drug resistance efflux transporter

WP_000999131.1 62 25 SAOUHSC_00058 quinolone resistance protein NorB

drug:proton antiporter WP_000038961.1 42 27 SAOUHSC_00078 siderophore biosynthesis protein SbnD multidrug MFS transporter WP_001820335.1 40 26 SAOUHSC_00952 glycolipid permease LtaA nitrate transporter NarT WP_000278558.1 52 24 SAOUHSC_02307 putative membrane spanning protein multidrug efflux MFS transporter NorA WP_001041272.1 84 20 SAOUHSC_02309 membrane protein chloramphenicol resistance protein DHA1 WP_000026194.1 41 20 SAOUHSC_02435 macrolide MFS transporter quinolone resistance protein NorB WP_001066546.1 34 23 SAOUHSC_02620 membrane protein conserved hypothetical protein ABD31642.1 62 25 SAOUHSC_02752 chloramphenicol resistance protein DHA1 hypothetical protein SAOUHSC_02435 YP_500904.1 34 22 SAOUHSC_02866 membrane protein hypothetical protein SAOUHSC_02752 YP_501211.1 51 20 ABD31816.1 membrane protein ABD31642.1 Bcr/CflA family drug resistance efflux transporter

QC, Query Cover; %ID, percentage of identity; MFS, major facilitator super-family MFS, major facilitator super-family Table 5: Physiochemical properties

Protein # AA MW pI # neg # pos EC II AI GRAVY

GlpT 451 50204.2 8.69 28 33 115660 36.12 99.73 0.505 LacY 417 46457.1 9.02 17 24 54240 29.54 109.93 0.906 EmrD 375 39995.1 9.10 12 18 61800 37.90 121.23 0.942 SAOUHSC_00058 462 48999.6 9.49 17 29 59610 22.37 128.74 0.948

SAOUHSC_00078 418 44835.3 9.54 13 27 53570 46.13 120.65 0.786

SAOUHSC_00952 402 45457.6 9.54 16 26 63830 36.90 128.28 0.936 SAOUHSC_02307 163 19404.9 9.62 16 22 32430 48.51 123.80 0.134 SAOUHSC_02309 159 18972.3 9.18 13 18 38850 36.78 108.43 0.140

SAOUHSC_02435 397 44337.3 9.44 16 31 47830 24.38 127.68 0.868

SAOUHSC_02620 215 24956.7 6.10 16 15 34630 56.72 111.91 0.573 SAOUHSC_02752 375 40245.1 9.68 12 20 43430 29.41 132.51 0.918 SAOUHSC_02866 822 90409.3 6.46 90 86 58790 31.15 107.93 0.159 ABD31816.1 416 47645.0 9.19 31 43 39810 26.46 107.62 0.282 ABD31642.1 312 34467.5 10.05 11 20 34950 28.19 125.61 0.897

# AA, number of amino acids; MW, molecular weight; pI, theoretical isoelectric point; # neg, total number of negatively charged residues (Asp + Glu); # pos, total number of positively charged residues (Arg + Lys); EC, extinction coefficient assuming all pairs of Cys residues form cystines; II, instability index; AI, aliphatic index; GRAVY, grand average hydropathy. Table 6: Transmembrane Regions Identified by SOSUI Locus Tag Number Base-Pair Position

GlpT 10 26 – 43, 102 – 124, 160 – 181, 187 – 208, 251 – 273, 290 – 312, 320 – 342, 350 – 372, 384 – 406, 414 – 435 LacY 12 11 – 33, 44 – 66, 75 – 97, 106 – 128, 144 – 166, 174 – 196, 215 – 237, 260 – 282, 288 – 310, 313 – 335, 346 – 368, 379 – 401 EmrD 10 4 – 26, 32 – 54, 69 – 91, 127 – 149, 153 – 175, 205 – 227, 235 – 256, 263 – 285, 291 – 313, 346 – 368 SAOUHSC_00058 13 12 – 34, 45 – 67, 88 – 110, 134 – 156, 163 – 184, 198 – 220, 226 – 247, 262 – 284, 301 – 323, 330 – 349, 355 – 373, 403 – 425, 431 – 453 SAOUHSC_00078 12 11 – 33, 45 – 67, 80 – 102, 104 – 126, 145 – 167, 171 – 192, 224 – 246, 256 – 277, 291 – 313, 317 – 339, 351 – 371, 376 – 398 SAOUHSC_ 00952 11 15 – 37, 42 – 64, 85 – 107, 113 – 134, 144 – 166, 173 – 195, 218 – 240, 251 – 273, 280 – 302, 307 – 329, 374 – 396 SAOUHSC_02307 2 17 – 39, 50 – 72 SAOUHSC_02309 2 20 – 42, 48 – 70 SAOUHSC_02435 11 5 – 27, 36 – 58, 66 – 88, 92 – 114, 133 – 155, 162 – 184, 212 – 234, 248 – 270, 286 – 308, 333 – 355, 361 – 382 SAOUHSC_02620 4 38 – 60, 73 – 95, 108 – 130, 143 – 165 SAOUHSC_02752 8 12 – 34, 53 – 75, 80 – 101, 110 – 132, 139 – 161, 193 – 215, 224 – 246, 260 – 282 SAOUHSC_02866 12 10 – 32, 170 – 192, 198 – 220, 225 – 246, 267 – 289, 303 – 324, 348 – 370, 510 – 532, 538 – 560, 579 – 601, 624 – 646, 655 – 677 ABD31816.1 6 75 – 97, 119 – 141, 143 – 165, 169 – 191, 195 – 216, 389 – 411 ABD31642.1 9 13 – 35, 53 – 74, 80 – 102, 107 – 129, 138 – 160, 167 – 189, 221 – 243, 254 – 276, 285 – 307

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Figure 4: Representative phylogenetic tree of proteins produced via PHYLIP package programs showing six hypothetical proteins belong evolutionarily to the major facilitator superfamily (MFS). SAOUHSC_00058, SAOUHSC_02435, SAOUHSC_02752 and ABD31642.1 are related to drug efflux proteins. SAOUHSC_00078 is closely related to a siderophore biosynthesis protein as SAOUHSC_00952 is confirmed to be a glycolipid permease. Table 5 lists the physiochemical parameters calculated by ExPASy. Since MFS proteins are usually 400 to 600 amino acids, any hypothetical protein outside the 300 to 700 amino acid range is unlikely to be functional. Four of the five hypothetical proteins that had ambiguous membrane protein BLASTP hits, SAOUHSC_02307, SAOUHSC_02309, SAOUHSC_02620, and SAOUHSC_02866, came up outside this size range. Examination of other physiochemical calculations failed to be consistent in predicting MFS proteins among hypotheticals though some proteins outside this size range had varied theoretical index (SAOUHSC_02620 and SAOUHSC_02866) and GRAVY values (<0.05 for SAOUHSC_02307, SAOUHSC_02309, SAOUHSC_02866, and ABD31816.1) compared to proteins with expected size. This

indicates that these specific hypothetical proteins may not be in the MFS. SOSUI calculates the average hydrophobicity of a protein. If hydrophobicity exists, the server labels that portion of the protein as a transmembrane region. Table 6 details transmembrane regions as described by the SOSUI server. MFS proteins typically have 11 to 13 transmembrane regions, so SAOUHSC_02307, SAOUHSC_02309, SAOUHSC_02620, and ABD31816.1 are unlikely functioning MFS proteins because they have half or less of the necessary number of transmembrane regions. Since GlpT and LacY had 10 transmembrane regions per SOSUI (data not shown), it is still possible that SAOUHSC_02752 and ABD31642.1 are related to MFS proteins based on this analysis.

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Table 7: CDD-BLAST domain data

Hypothetical Protein Domains

cd06174 GlpT PRK11273 cd06174 LacY pfam01306 cd06174 EmrD PRK11652 cd06174 SAOUHSC_00058 pfam07690 cd06174 SAOUHSC_00078 PRK09874 cd06174 SAOUHSC_00952 pfam07690

SAOUHSC_02307 COG3402 SAOUHSC_02309 cl01348

cd06174 SAOUHSC_02435 TIGR00900

SAOUHSC_02620 COG3152 cd06174 SAOUHSC_02752 COG2814 pfam03176 cl21543

SAOUHSC_02866

COG2409 ABD31816.1 COG1289 ABD31642.1 cd06174

cd06174 and pfam07690, Major Facilitator Superfamily; PRK11273, glycerol-3-phosphate transporter; pfam01306, LacY proton/sugar symporter; PRK11652, multidrug resistance protein D; PRK09874, drug efflux system protein MdtG; COG3402, YdbS; cl01348, bPH_2 super family; TIGR00900, H+ Antiporter protein; COG3152, yhaH; COG2814, arabinose efflux permease; pfam03176 and cl21543, MMPL super family; COG2409, YdfJ; COG1289, YccC. Finally, potential MFS hypothetical proteins should have similar domains to those found in GlpT, LacY, and EmrD. Table 7 lists the CDD-BLAST results and Table 8 the Pfam results for domain identification. Most hypothetical proteins with the appropriate size and well-defined PSI-BLAST homologs had the CD06174 MFS domain identified by CDD-BLAST and the MFS_1 domain identified by Pfam. GlpT and EmrD have these domains too. Based on these data collectively, the following hypothetical proteins are likely MFS proteins due to their size (300 – 700 amino acids), well-defined BLAST homologs (no generalized membrane or hypothetical proteins), more than seven transmembrane regions, and CD06174 and MFS domains from CDD-BLAST and Pfam, respectively, underwent evolutionary analyses: SAOUHSC_00058, SAOUHSC_00078, SAOUHSC_00952, SAOUHSC_02435, SAOUHSC_02752, and ABD31642.1. Since either the GlpT or EmrD proteins identified the six hypothetical proteins most likely to belong to the MFS, the study removed LacY and its homologs in Table 2 from further study.

Table 8: Pfam domain data

Hypothetical Protein Domains

GlpT MFS_1 LacY LacY_symp EmrD MFS_1 SAOUHSC_00058 MFS_1

MFS_1 SAOUHSC_00078 sugar_tr

SAOUHSC_00952 MFS_1 SAOUHSC_02307 bPH_2 SAOUHSC_02309 No Pfam-A matches SAOUHSC_02435 MFS_3 SAOUHSC_02620 DUF805 SAOUHSC_02752 MFS_1 SAOUHSC_02866 MMPL family ABD31816.1 No Pfam-A matches ABD31642.1 MFS_1

MFS_1, Major Facilitator Superfamily; LacY_symp, LacY proton/sugar symporter; sugar_tr, sugar and other transporter; bPH_2, bacterial Pleckstrin homology domain; MFS_3, Transmembrane secretion effector; DUF805, Protein of unknown function; MMPL family, putative integral membrane proteins. To evaluate how related these hypothetical proteins were, proteins of interest underwent multiple sequence alignment and phylogenetic tree construction. For these analyses, all defined homologs from Tables 1 and 3 combined with hypothetical proteins fitting the study’s criteria completed multiple sequence alignment as displayed in Figures 2 and 3, respectively. These analyses included top BLASTP hits from Table 4 not already included in Tables 1 and 3. Both alignments found over 10 alpha helices in the consensus sequences, as expected from MFS members. Hypothetical proteins aligned with their top BLASTP hits from Table 4 best, with SAOUHSC_00952 also closely aligning with the MFS transporter. To visualize how closely related these proteins are phylogenetic trees were constructed. Though all similar, a representative tree of all the proteins and their homologs is shown in Figure 4. The same NorA multidrug efflux MFS transporter came up in all three E. coli PSI-BLASTs while GlpT (1PW4) and EmrD (3GFP) identified two separate NorB quinolone resistance proteins. SAOUHSC_00058 nuzzled between the two NorB proteins. As expected, phylogeny confirmed the multiple sequence alignment. Hypothetical proteins related closely with their top PSI-BLAST hits from Table 4, with SAOUHSC_00952 being closely related to the MFS transporter also. Floyd’s illustration of the established proteins shown in the phylogenetic tree presented here is similarly arranged [4].

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Conclusion: This study identified eleven hypothetical proteins homologous to E. coli MFS proteins with known structure. Six of those hypothetical proteins, SAOUHSC_00058, SAOUHSC_00078, SAOUHSC_00952, SAOUHSC_02435, SAOUHSC_02752, and ABD31642.1, were between 300 and 700 amino acids, had well-defined BLASTP homologs, over seven transmembrane regions, CD06174 domain from CDD-BLAST, and an MFS domain from Pfam. Based on these results alongside multiple sequence alignment and phylogenetic trees, SAOUHSC_00058 is likely a quinolone resistance protein (NorB) due to its close relation to two NorB proteins identified by either GlpT or EmrD. SAOUHSC_00058 may be a siderophore biosynthesis protein (SbnD). SAOUHSC_00952, a glycolipid permease (LtaA), and another MFS transporter closely are related. Further, UniProt has SAOUHSC_00952 labeled as a glycolipid permease LtaA with experimental evidence at the protein level, unlike all the other hypothetical proteins studied here labeled as uncharacterized [29]. SAOUHSC_02435 hits a macrolide MFS transporter while SAOUHSC_02752 matches a chloramphenicol resistance (DHA1), and ABD31642.1 may be a Bcr/CflA family drug resistance efflux transporter. These data manually verifies the in silico identity of six hypothetical proteins from reference strain S. aureus NCTC 8325 in public databases. References: [1] Holden MT et al. Proc. Natl. Acad. Sci. U. S. A. 2004 101:26

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Edited by P Kangueane

Citation: Marklevitz & Harris, Bioinformation 12(4): 254-262 (2016)

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